Mycobacteria infections remain a major problem in human health. Mycobacterium tuberculosis infects approximately 1/3 of the world's population; Mycobacterium leprae (ML) is an endemic infection in many parts of the world. M. marinum (Mm) is a natural pathogen of fish and can cause chronic disease in frogs with many features of tuberculosis. Because of this, its rapid growth in vitro, its suitability for forward genetics, its minimal pathogenicity for laboratory" workers, and its ability to grow in mammalian macrophages in vitro, Mm can be a facile model to discover genes involved in pathogenesis of Mycobacteria infections. We have developed a transposon mutagenesis plasmid for use in Mm that has proven to provide an excellent library of random mutations. From that random insertion library, we have screened >1000 mutants for ability to grow in macrophages and have characterized >25 mutants that fail to grow normally. For each mutant, we have sequenced the transposon insertion site, and, using this information, we have discovered several genes required for intracellular growth of Mm. We propose to focus on a more detailed characterization of two most interesting mutants to understand the roles of the targeted genes in host cell invasion by, and intracellular growth of, Mycobacteria. In each case, we have shown that the Mtb homologue of the targeted Mm genes will complement the phenotypic defects discovered in the mutant. This allows a rapid and thorough investigation of the roles of these Mtb genes in a model that is capable of intracellular growth and ultimately of extension to in vivo models of infection. The purpose of the current proposal is to use these mutants to develop a better understanding of Mycobacteria entry into and survival in macrophages, and of macrophage response to Mycobacteria infection, critical events in the pathogenesis of disease. Our specific aims are: 1. Characterize the mip locus, required by Mm for host cell invasion and intracellular survival in macrophages. 2. Determine how the GDP-mannose synthesis operon regulates macrophage activation and intracellular survival. 3. Carry out a genetic screen for Mm genes specifically required for intracellular growth in macrophages.